Methods of making chitosan

Abstract

A method for making a chitosan product from yeast cells is disclosed herein. Yeast cells are cultured to form a biomass of yeast cells. The yeast cells are induced to undergo meiosis causing the yeast cells to form asci containing ascospores wherein each ascospore contains a chitosan protective layer in the ascospore wall. Chitosan is extracted from the ascospores, purified to form purified chitosan, and precipitated and dried to form a chitosan product.

1 - 28 . (canceled) 
     
     
         29 . A method of making a chitosan product, comprising:
 culturing yeast cells, wherein the cultured yeast cells form a biomass of yeast cells;   inducing the cultured yeast cells to undergo meiosis, wherein the induced yeast cells form asci with ascospores and wherein each ascospore contains a chitosan protective layer in an ascospore wall;   extracting chitosan from the ascospore walls;   purifying the chitosan to form purified chitosan; and   precipitating and drying the purified chitosan to form a chitosan product.   
     
     
         30 . The method of  claim 29 , wherein the method further includes an additional step selected from the group consisting of: disrupting asci walls or disrupting the asci walls and the ascospore walls after inducing the cultured yeast cells to undergo meiosis; disrupting the asci walls and separating the ascospores from debris after inducing the cultured yeast cells to undergo meiosis; and disrupting the ascospore walls after separating the ascospores from the debris. 
     
     
         31 . The method of  claim 29 , wherein the yeast cells are a yeast selected from the group consisting of an Ascomycota phylum yeast strain, a Saccharomycotina subphyla yeast strain, a class Saccharomycetes yeast strain, a Genus  Saccharomyces  yeast strain, a Genus  Komagataella  yeast strain, a Genus  Schizosaccharomyces  yeast strain, a heterothallic haploid yeast strain, a heterothallic diploid yeast strain, and a heterothallic polyploid yeast strain. 
     
     
         32 . The method of  claim 31 , wherein the yeast are sporulating yeast cells that can be fused to each other to form hybrid cells. 
     
     
         33 . The method of  claim 31 , wherein the yeast are heterothallic yeast strains of opposite mating types that can mate with each other to form heterozygous mating type hybrid cells. 
     
     
         34 . The method of  claim 29 , wherein the yeast is selected from the group consisting of a homothallic self-fertile yeast strain, a heterothallic heterozygous for mating type diploid yeast strain, a heterothallic heterozygous for mating type polyploid yeast strain, and combinations thereof. 
     
     
         35 . The method of  claim 34 , wherein cells of homothallic self-fertile yeast strain, the heterothallic heterozygous for mating type diploid yeast strain, a heterothallic heterozygous for mating type polyploid yeast strain or combinations thereof produce ascospores. 
     
     
         36 . The method of  claim 29 , wherein inducing the cultured yeast cells to undergo meiosis includes:
 incubating the yeast cells in a presporulation medium for a time ranging from about 6 hours to about 72 hours; and   transferring the yeast cells to a sporulation medium and incubating the yeast cells for a time ranging from about 12 hours to about 144 hours, thereby converting about 10% to about 100% of the biomass of yeast cells to asci containing at least one ascospore.   
     
     
         37 . The method of  claim 29 , wherein inducing the cultured yeast cells to undergo meiosis includes transferring the yeast cells to a sporulation medium and incubating the yeast cells for a time ranging from about 12 hours to about 144 hours, thereby converting about 10% to about 100% of the yeast cells to asci containing at least one ascospore. 
     
     
         38 . The method of  claim 29 , wherein culturing the yeast cells is performed using a system selected from the group consisting of a cell culture vessel, a cell retention system, and a media exchange system. 
     
     
         39 . The method of  claim 30 , wherein disrupting the asci walls or disrupting the asci walls and ascospore walls is performed enzymatically or mechanically. 
     
     
         40 . The method of  claim 30 , wherein separating the ascospores from the debris includes using density gradient centrifugation or biphasic separation. 
     
     
         41 . The method of  claim 40 , wherein a density gradient is created using a density gradient medium selected from the group consisting of polyhydric alcohols, polysaccharides, colloidal silica, and combinations thereof. 
     
     
         42 . The method of  claim 40 , wherein biphasic separation includes:
 suspending the ascospores and debris into a buffer solution or an aqueous medium, thereby forming a suspension;   mixing the suspension with a solution having a different phase from the suspension for a time ranging from about 1 second to about 1800 seconds;   partitioning the ascospores into an ascospore containing chitosan layer; and   retaining the ascospore containing chitosan layer.   
     
     
         43 . The method of  claim 40 , wherein the biphasic separation is an aqueous two phase extraction including mixing the ascospores and the debris with an intermediate molecular weight polyether in an aqueous salt solution to partition the ascospores into a polyether phase. 
     
     
         44 . The method of  claim 29 , wherein extracting chitosan from the ascospore walls includes heating the ascospores in an acid solution to a temperature ranging from about 60° C. to about 100° C. for at time ranging from about 1 hour to about 12 hours, thereby solubilizing the chitosan. 
     
     
         45 . The method of  claim 44 , wherein the acid solution is present in an amount ranging from about 0.1% w/v to about 50% w/v of a total weight of the acid solution. 
     
     
         46 . The method of  claim 29 , wherein precipitating and drying the purified chitosan includes:
 washing the chitosan with an alkaline solution, thereby precipitating the chitosan; and   drying the chitosan to form the chitosan product.   
     
     
         47 . The method of  claim 46 , wherein the alkaline solution is a solution of sodium hydroxide or ammonium hydroxide present in an amount ranging from about 3% w/v and about 30% w/v of a total weight of the alkaline solution. 
     
     
         48 . The method of  claim 47 , wherein purifying the chitosan includes enzymatic digestion by adding an enzyme to the acid solution at a temperature ranging from about 4° C. to about 105° C. for a time ranging from about 1 hours to about 24 hours.